Early-life nicotine or cotinine exposure produces long-lasting sleep alterations and downregulation of hippocampal corticosteroid receptors in adult mice.

Early-life exposure to environmental toxins like tobacco can permanently re-program body structure and function. Here, we investigated the long-term effects on mouse adult sleep phenotype exerted by early-life exposure to nicotine or to its principal metabolite, cotinine. Moreover, we investigated whether these effects occurred together with a reprogramming of the activity of the hippocampus, a key structure to coordinate the hormonal stress response. Adult male mice born from dams subjected to nicotine (NIC), cotinine (COT) or vehicle (CTRL) treatment in drinking water were implanted with electrodes for sleep recordings. NIC and COT mice spent significantly more time awake than CTRL mice at the transition between the rest (light) and the activity (dark) period. NIC and COT mice showed hippocampal glucocorticoid receptor (GR) downregulation compared to CTRL mice, and NIC mice also showed hippocampal mineralocorticoid receptor downregulation. Hippocampal GR expression significantly and inversely correlated with the amount of wakefulness at the light-to-dark transition, while no changes in DNA methylation were found. We demonstrated that early-life exposure to nicotine (and cotinine) concomitantly entails long-lasting reprogramming of hippocampal activity and sleep phenotype suggesting that the adult sleep phenotype may be modulated by events that occurred during that critical period of life.

Structural binding perspectives of a major tobacco alkaloid, nicotine, and its metabolite cotinine with sex-steroid nuclear receptors.

Globally, more than a billion people smoke tobacco making it one of the biggest public health problems and a leading risk factor for global deaths. Nicotine, the main alkaloid in tobacco, has been shown to be associated with fertility problems in men and women. The adverse effects of tobacco/nicotine on reproduction have been attributed to deleterious effects on gametes, steroidogenic imbalance, and competitive inhibition of steroid receptors. The present study reports the sex-steroid receptor disrupting potential of nicotine and its major metabolite cotinine against the estrogen receptor-α (ERα), ERß, androgen receptor (AR), and progesterone receptor (PR). Both ligands bound in the ligand-binding pockets of ERα, ERß, AR and PR and formed important hydrophobic interactions with different amino-acid residues of receptors. Most of the residues of ERα, ERß, AR and PR interacting with nicotine and cotinine were common with those of native/bound ligands of the receptors. Interacting amino acids most important for binding of nicotine and cotinine with each receptor were identified by loss in accessible surface area. Amino acids Leucine-346, Leucine-384 and Phenylalanine-404 for ERα; Methionine-336, Phenylalanine-356 and Leucine-298 for ERß; and Leucine-704 and Leucine-718, respectively for AR and PR, were the most important residues for binding with nicotine and cotinine. Among the four receptors, based on the number of interactions, nicotine and cotinine had greater potential to interfere in the signaling of ERß. In conclusion, the results suggested that nicotine and cotinine bind and interact with sex-steroid nuclear receptors and have potential to interfere in the steroid hormone signaling resulting in reproductive dysfunction.

Short-Term Exposure to Waterpipe/Hookah Smoke Triggers a Hyperactive Platelet Activation State and Increases the Risk of Thrombogenesis.

OBJECTIVE: Cardiovascular disease is a major public health problem. Among cardiovascular disease's risk factors, tobacco smoking is considered the single most preventable cause of death, with thrombosis being the main mechanism of cardiovascular disease mortality in smokers. While tobacco smoking has been on the decline, the use of waterpipes/hookah has been rising, mainly due to the perception that they are less harmful than regular cigarettes. Strikingly, there are few studies on the negative effects of waterpipes on the cardiovascular system, and none regarding their direct contribution to thrombus formation. Approach and Results: We used a waterpipe whole-body exposure protocol that mimics real-life human exposure scenarios and investigated its effects, relative to clean air, on platelet function, hemostasis, and thrombogenesis. We found that waterpipe smoke (WPS)-exposed mice exhibited both shortened thrombus occlusion and bleeding times. Further, our results show that platelets from WPS-exposed mice are hyperactive, with enhanced agonist-induced aggregation, dense and α-granule secretion, αIIbß3 integrin activation, phosphatidylserine expression, and platelet spreading, when compared with clean air-exposed platelets. Finally, at the molecular level, it was found that Akt (protein kinase B) and ERK (extracellular signal-regulated kinases) phosphorylation are enhanced in the WPS and in nicotine-treated platelets. CONCLUSIONS: Our findings demonstrate that WPS exposure directly modulates hemostasis and increases the risk of thrombosis and that this is mediated, in part, via a state of platelet hyperactivity. The negative health impact of WPS/hookah, therefore, should not be underestimated. Moreover, this study should also help in raising public awareness of the toxic effects of waterpipe/hookah.

Cotinine, a major nicotine metabolite, induces cell proliferation on urothelium in vitro and in vivo.

Tobacco smoking is a major risk factor for human cancers including urinary bladder carcinoma. In a previous study, nicotine enhanced rat urinary bladder carcinogenesis using a rat urinary bladder two-stage carcinogenesis model. In the present study, nicotine metabolites (cotinine, trans-3'-hydroxy cotinine and N'-nitrosonornicotine) were evaluated in a cell proliferation assay using urinary bladder urothelial cell lines. Cotinine (0.1 to 1 mM) induced the highest cell proliferation compared to the others, including nicotine, in three bladder cancer cell lines (RT4, T24 and UMUC3 cells). By Western blot, cotinine induced phosphorylation of Stat3 and expression of cyclin D1 in UMUC3 cells. The cell proliferation induced by cotinine was blocked by inhibitors of nicotinic receptors (10 nM SR16584 or 10 µM methyllycaconitine citrate) and Stat3 (100 nM stattic). In an in vivo study, cotinine (13, 40 and 120 ppm) in drinking water also induced cell proliferation and simple hyperplasia in urinary bladder and renal pelvis urothelium of rats, but to a lesser degree compared to nicotine (40 ppm). Cytotoxicity detected by scanning electron microscopy and apoptosis in the bladder urothelium were induced by nicotine but not cotinine. These data suggest that cotinine is able to induce urothelial cell proliferation both in vitro and in vivo, and high urinary concentrations may enhance urothelial carcinogenesis.

Preliminary evidence from planarians that cotinine establishes a conditioned place preference.

While the psychoactive stimulant nicotine has been the subject of extensive research, considerably less attention has focused on other compounds found in either tobacco smoke or that are nicotine metabolites. Recent papers have suggested that some of the compounds in question may either alter nicotine's effects or have reinforcing properties themselves, although they would only be experienced after consumption of tobacco. The potential for these compounds to function as reinforcers or to potentiate the reinforcing properties of nicotine merits investigation. To pursue this line of inquiry, we examined cotinine in a planarian model of environmental place preference. In the present study, planarians demonstrated that the compound cotinine, which is present in tobacco smoke, and is also the principal nicotine metabolite, establishes a conditioned place preference. These data represent the first ever demonstration that cotinine will establish a conditioned place preference in planarians and possibly contribute to the addictive properties of nicotine.

Efficacy of vitamins E and C for reversing the cytotoxic effects of nicotine and cotinine.

Nicotine has adverse cellular and molecular effects on oral mucosa, bone, and teeth. Vitamin E (α-tocopherol) and vitamin C (ascorbic acid) are biological antioxidants with positive effects on wound healing and bone formation. This in vitro study sought to assess the cytotoxic effects of different concentrations of nicotine and cotinine (a metabolite of nicotine) on MG-63 osteoblast-like cells and human gingival fibroblasts (HGFs) in the presence and absence of antioxidant vitamins E and C (separately and combined). Cell viability and proliferation were assessed using the methyl thiazol tetrazolium (MTT) assay. Cell migration was assessed using the scratch test, and expression of apoptosis-related genes was quantitatively analyzed using real-time PCR. Dose-dependent negative effects of nicotine on the morphology, viability, proliferation, and migration of MG-63 and HGF cells were statistically significantly greater than those of cotinine. Vitamin E (separately and combined with vitamin C) was statistically significantly more effective than vitamin C (at the concentration used in this study) at improving cell viability, proliferation, and migration, and at reducing apoptosis of cells exposed to nicotine or cotinine. Based on the positive results of this study, vitamin C and especially vitamin E (systemically and/or locally) may be useful in the repair and regeneration of oral hard and soft tissues in smokers.

Age-dependent sensitivity of the mouse kidney to chronic nicotine exposure.

BackgroundMany adolescents are exposed to nicotine via smoking, e-cigarette use, or second-hand smoke. Nicotine-induced renal oxidative stress and its long-term consequences may be higher in adolescents than in adults because of intrinsic factors in the adolescent kidney.MethodsAdolescent and adult male C57Bl/6J mice were subjected to 2 or 200 µg/ml nicotine, which closely emulates passive or active smoking, respectively, for 4 weeks. Extent of nicotine exposure (cotinine content), oxidative stress (HNE), renal function (creatinine), tubular injury (KIM-1), and pretreatment renal levels of select pro-oxidant (p66shc) and antioxidant (Nrf2/MnSOD) genes were determined. Impact of p66shc overexpression or Nrf2/MnSOD knockdown on low-/high-dose nicotine-induced oxidative stress was determined in cultured renal proximal tubule cells.ResultsDespite similar plasma/renal cotinine levels, renal HNE and KIM-1 contents were higher in adolescents compared with those in adults, whereas renal function was unaltered after passive or active smoking-equivalent nicotine exposure. Pretreatment levels of p66shc were higher, whereas Nrf2/MnSOD levels were lower in the adolescent kidney. In agreement with this, overexpression of p66shc or knockdown of Nrf2/MnSOD augmented nicotine-induced ROS production in renal proximal tubule cells.ConclusionChronic nicotine exposure incites higher oxidative stress in the adolescent than in adult kidney because of a pre-existent pro-oxidant milieu.

Modelling foetal exposure to maternal smoking using hepatoblasts from pluripotent stem cells.

The liver is a dynamic organ which is both multifunctional and highly regenerative. A major role of the liver is to process both endo and xenobiotics. Cigarettes are an example of a legal and widely used drug which can cause major health problems for adults and constitute a particular risk to the foetus, if the mother smokes during pregnancy. Cigarette smoke contains a complex mixture of thousands of different xenobiotics, including nicotine and polycyclic aromatic hydrocarbons. These affect foetal development in a sex-specific manner, inducing sex-dependant molecular responses in different organs. To date, the effect of maternal smoking on the foetal liver has been studied in vitro using cell lines, primary tissue and animal models. While these models have proven to be useful, poor cell phenotype, tissue scarcity, batch-to-batch variation and species differences have led to difficulties in data extrapolation toward human development. Therefore, in this study we have employed hepatoblasts, derived from pluripotent stem cells, to model the effects of xenobiotics from cigarette smoke on human hepatocyte development. Highly pure hepatocyte populations (>90%) were produced in vitro and exposed to factors present in cigarette smoke. Analysis of ATP levels revealed that, independent of the sex, the majority of smoking derivatives tested individually did not deplete ATP levels below 50%. However, following exposure to a cocktail of smoking derivatives, ATP production fell below 50% in a sex-dependent manner. This was paralleled by a loss metabolic activity and secretory ability in both female and male hepatocytes. Interestingly, cell depletion was less pronounced in female hepatocytes, whereas caspase activation was ~twofold greater, indicating sex differences in cell death upon exposure to the smoking derivatives tested.

Nicotine and Cotinine Levels With Electronic Cigarette: A Review.

BACKGROUND: Since their introduction in 2004, electronic cigarettes (e-cigarettes) have gained popularity worldwide. E-cigarettes are marketed as nicotine delivery devices. Commonly reported reasons for use include to quit smoking, to reduce urge to smoke, or the perceived lower risk alternative to smoking. But what are the actual amounts of nicotine delivered? AIM: This review summarizes all the published studies concerning nicotine or cotinine levels following e-cigarette use. METHODS: A literature search was conducted from the PubMed database, from 1985 to January 2014, using the following terms: electronic cigarette(s), e-cigarette(s), electronic nicotine delivery system, cotinine, and nicotine. Articles were excluded if they were not pertinent according to our criteria. References of all relevant articles were also evaluated. RESULTS: Eight studies were included in this review. The following information was extracted from the articles: population size, age of participants, recruitment, inclusion and exclusion criteria, concentration of nicotine in refills liquids, study sample design, and observed concentrations. Following design of studies, plasma nicotine Cmax was observed between 0 and 5 ng/mL (no significant changes) or between 13.9 and 16.3 ng/mL (similar to a tobacco cigarette) with a Tmax between 70 and 75 minutes. Cotinine levels after "vaping" an e-cigarette are similar to a tobacco cigarette. CONCLUSION: This review summarizes e-cigarette studies that contain information on nicotine or cotinine levels. The peak concentration of nicotine appears to be dependent on the use and dose level of e-cigarette cartridge. The value of this peak concentration is similar to the value found with a tobacco cigarette. However, the time corresponding to the peak concentration is delayed compared to a tobacco cigarette.

Cotinina urinaria en embarazadas expuestas al humo ambiental del tabaco que concurren a controles prenatales en la ciudad de Gualeguaychú / Urinary cotinine values and exposure to environmental snuff in pregnant women who attend prenatal examinations in the city of Gualeguaychú

El tabaco es uno de los factores de riesgo prevenibles más importante de las enfermedades crónicas no trasmisibles (ECNT). Los efectos de fumar no están limitados a los fumadores activos, involucran además a los individuos que sufren los efectos de los tóxicos del humo del tabaco ambiental (HTA): los fumadores pasivos. Las mujeres embarazadas fumadoras activas o expuestas al HTA son una población muy sensible a los efectos tóxicos del tabaco, ya que los mismos repercuten también sobre el feto en formación. La cotinina es en la actualidad el marcador biológico más adecuado para medir exposición al HTA tanto activa como pasiva. Objetivo: determinar el nivel de cotinina en mujeres embarazadas que manifestaron ser fumadoras pasivas, relacionando los valores obtenidos con los días de exposición manifestados. Materiales y métodos: se trabajó con 443 embarazadas que concurrieron a centros de salud públicos y a un centro privado de Gualeguaychú para su control prenatal, solicitándoles a las que manifestaron estar expuestas al HTA una muestra de orina para el dosaje de cotinina. Se aplicó un diseño de tipo no experimental, retrospectivo y de corte transversal. El dosaje de cotinina se realizó en orina, empleando una metodología quimioluminiscente. Previamente se obtuvo un valor referencial de cotinina urinaria inferior a los 15,2 ng/ml para el 98 % de sujetos no fumadores no expuestos al HTA. Resultados: los análisis de los niveles de cotinina en las embarazadas fumadoras pasivas revelaron que, el 82 % en los centros públicos y el 42 % en el centro privado, presentaron un nivel de cotinina superior a 15,2 ng/ml. Teniendo en cuenta los días de exposición, el registro promedio de cotinina para las que manifestaron estar expuestas los últimos siete días fue de 52,3 ng/ml en el sector público y 64,1 ng/ml en el privado. Discusión y conclusiones: la medición de cotinina resulta de utilidad para tener datos fidedignos de la exposición pasiva al HTA. En los centros públicos el 82 % de las embarazadas que manifestaron estar expuestas tenían valores de cotinina urinaria que coincidían con lo expresado, mientras que en el centro privado el 42 % de las que manifestaron la misma situación presentaba valores del indicador que denotaban exposición al tabaco. Se observó un aumento progresivo del promedio del indicador biológico de acuerdo a los días de exposición en ambos sectores, superando los 50ng/ml cuando la exposición declarada fue durante los últimos 7 días, lo que es indicativo de una exposición al HTA severa. El interés y preocupación manifestados por las embarazadas que participaron en este estudio indica que la implementación de este tipo de diagnóstico puede contribuir a las campañas de prevención contra el consumo de tabaco y promover el derecho de quienes no fuman a vivir en ambientes saludables libres de los compuestos tóxicos del mismo.

Tobacco is one of the preventable risk factors, which is most important in the chronic non-communicable diseases (NCDs). The effects of smoking are not limited to active smokers; it also involves individuals who suffer the effects of environmental tobacco smoke (ETS): passive smokers. Pregnant women who are active smokers or exposed to ETS are a very sensitive population to the toxic effects of snuff, since they also affect the developing fetus. Cotinine is currently the most suitable biomarker for measuring ETS exposure both active and passive. Objective: To determine the level of cotinine in pregnant women who reported being passive smokers, relating the values obtained with the indicated days of exposure. Materials and methods: We worked with 443 pregnant women attending public health centers and a private centre in Gualeguaychú for prenatal care, asking to be exposed to ETS showed a urine sample for cotinine dosage. We performed a non-experimental, retrospective and cross-sectional design. The dosage of cotinine in urine was performed using a chemiluminescent method. Previously we obtained a reference value of urinary cotinine less than 15,2ng/ml for 98% of non smokers unexposed to ETS. Results: The analysis of cotinine levels in passive smoking pregnant women show tHTA in public centers, 82% has a cotinine level greater than 15,2ng/embaml, whereas in the private centre, 42% have the same range values. Considering the days of exposure, the average cotinine log for those who said were exposed for the past seven days, was 52.32 ng/ml in the public sector and 64.17 ng/ml in the private one. Discussion and conclusion: The measurement of cotinine is useful to have reliable data from passive exposure to ETS. In public centers, 82% of pregnant women who said were exposed had urinary cotinine levels consistent with the statement, while in the private centre the 42% who said had the same situation had indicator values denoting exposure to snuff. There was a progressive increase in average biological indicator according to the days of exposure in both sectors, exceeding 50ng/ml when the declared exposure was during the last 7 days, which is indicative of a severe ETS exposure. The interest and concern expressed by the pregnant women who participated in this study indicates HTA the implementation of this kind of diagnosis may contribute to prevention campaigns against snuff consumption and promote the right of nonsmokers to live in healthy environments free of the toxic compounds thereof.

Effects of nicotine, its metabolites and tobacco extracts on human platelet function in vitro.

Cigarette smoking is a leading cause of cardiovascular disease. The cardiovascular effects of smoking are probably multifactorial, including effects on platelets. Previous reports investigating the effects of nicotine and tobacco on platelet function are inconsistent. The present study investigated in vitro effects of nicotine, its major metabolites, tobacco extracts and extract of tobacco-free snuff on human platelets. None of the metabolites cotinine, cotinine-N-oxide, nicotine-1'-N-oxide or trans-3'-hydroxycotinine (0.1-10 µM) affected platelet aggregation or P-selectin expression. Nicotine (10 µM) weakly increased platelet aggregation, whereas trans-3'-hydroxycotinine (0.1 µM) and nicotine-1'-N-oxide (1-10 µM) weakly inhibited adhesion to fibrinogen. To elucidate the influence of other tobacco compounds, we investigated the impact of moist tobacco and smoke extracts on platelet function. Filtered extracts of oral snuff, cigarette smoke and tobacco free snuff inhibited platelet adhesion concentration-dependently. The inhibitory effects of tobacco extracts on platelet adhesion were independent of nicotine content and the nitric-oxide-pathway and not mediated through a platelet-nicotine-receptor. Taken together, tobacco extracts inhibit platelet activation during short-term in vitro challenge. As only limited effects of nicotine and nicotine metabolites were seen, the tobacco-induced platelet inhibition are likely induced by other compounds present in tobacco and tobacco free snuff.

Influence of nicotine and cotinine impregnation on the first step of periodontal regeneration: clot stabilization.

This study analyzes the clot stabilization on root surfaces of teeth impregnated with cotinine and nicotine and the influence of the scaling in the adhesion of blood components, observing the influence of new exposition to nicotine and/or cotinine after scaling. Fifteen human teeth extracted due to periodontal disease of non-smokers patients were selected and manually scaled. Four dentin blocks were obtained from each tooth (n = 60). Samples received blood application or reimpregnation with nicotine and/or cotinine, depending on the groups. Group 1: PBS immersion + root scaling + blood; group 2: nicotine + root scaling + blood; group 3: nicotine + root scaling + nicotine reapplication + blood; group 4: cotinine + root scaling + blood; group 5: cotinine + root scaling + cotinine reapplication+ blood; group 6: nicotine and cotinine + root scaling + nicotine and cotinine + blood. Samples were kept in 2 ml of each substance for 24 hours. Each group received a blood drop and was analyzed by SEM. The higher amount of blood components was present in teeth exposed to cotinine and the groups submitted to scaling and blood application in comparison with groups that received reapplication of toxic substances after scaling. The greater toxic effect on root dentin surface was after the exposure to nicotine and cotinine. Results suggest that periodontal healing may be delayed in smokers due to the direct inhibition of clot stabilization on the root surface when nicotine and cotinine are present concomitantly.

Lung tumorigenesis promoted by anti-apoptotic effects of cotinine, a nicotine metabolite through activation of PI3K/Akt pathway.

We previously found that genetic polymorphism in cytochrome P450 2A6 (CYP2A6) is one of the potential determinants of tobacco-related lung cancer risk. It has been reported that the plasma concentration of cotinine, a major metabolite of nicotine, in carriers of wild-type alleles of CYP2A6 is considerably higher than that in carriers of null or reduced-function alleles of CYP2A6, raising the possibility that cotinine plays an important role in the development of lung cancer. As a novel mechanism of lung tumorigenesis mediated by CYP2A6, we investigated the effects of cotinine on the suppression of apoptosis and promotion of lung tumor growth. In human lung adenocarcinoma A549 cells, cotinine inhibited doxorubicin-induced cell death by suppressing caspase-mediated apoptosis. Enhanced phosphorylation of Akt, a key factor responsible for cell survival and inhibition of apoptosis, was detected after cotinine treatment. These data suggest that cotinine suppresses caspase-mediated apoptosis induced by doxorubicin through activation of the PI3K/Akt pathway. Furthermore, we clarified that cotinine significantly facilitated tumor growth in the Lewis lung cancer model and accelerated development of lung adenomas induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mice. We herein propose that cotinine induces tumor promotion by inhibiting apoptosis and enhancing cellular proliferation, thus underlining the importance of CYP2A6 in tobacco-related lung tumorigenesis.

Thirdhand tobacco smoke: emerging evidence and arguments for a multidisciplinary research agenda.

BACKGROUND: There is broad consensus regarding the health impact of tobacco use and secondhand smoke exposure, yet considerable ambiguity exists about the nature and consequences of thirdhand smoke (THS). OBJECTIVES: We introduce definitions of THS and THS exposure and review recent findings about constituents, indoor sorption-desorption dynamics, and transformations of THS; distribution and persistence of THS in residential settings; implications for pathways of exposure; potential clinical significance and health effects; and behavioral and policy issues that affect and are affected by THS. DISCUSSION: Physical and chemical transformations of tobacco smoke pollutants take place over time scales ranging from seconds to months and include the creation of secondary pollutants that in some cases are more toxic (e.g., tobacco-specific nitrosamines). THS persists in real-world residential settings in the air, dust, and surfaces and is associated with elevated levels of nicotine on hands and cotinine in urine of nonsmokers residing in homes previously occupied by smokers. Much still needs to be learned about the chemistry, exposure, toxicology, health risks, and policy implications of THS. CONCLUSION: The existing evidence on THS provides strong support for pursuing a programmatic research agenda to close gaps in our current understanding of the chemistry, exposure, toxicology, and health effects of THS, as well as its behavioral, economic, and sociocultural considerations and consequences. Such a research agenda is necessary to illuminate the role of THS in existing and future tobacco control efforts to decrease smoking initiation and smoking levels, to increase cessation attempts and sustained cessation, and to reduce the cumulative effects of tobacco use on morbidity and mortality.

Pharmacokinetic study of nicotine and its metabolite cotinine to clarify possible association between smoking and voiding dysfunction in rats using UPLC/ESI-MS.

The present study was undertaken to clarify the possible association between nicotine intake/cigarette smoking and detrusor instability. For pharmacokinetic characterization of nicotine and cotinine (a major and pharmacologically less active metabolite of nicotine), a rapid ultra-performance liquid chromatography/electrospray ionization-mass spectrometry (UPLC/ESI-MS) method was developed that requires only a small amount of sample and simple pretreatment. The UPLC/ESI-MS method was validated with a focus on specificity, sensitivity (limit of detection, 2.5 ng/mL; limit of quantification, 5 ng/mL), linearity (r > 0.998), accuracy (97.2-102.8%), precision (relative standard deviation <8%) and robustness in accordance with ICH guidelines (Q2B Validation of Analytical Procedures: Methodology). The developed method was successfully applied to determine nicotine and cotinine levels in rat biological samples such as plasma, urine and several tissues. After subcutaneous administration of nicotine ditartrate (2 mg/kg of body weight) in rats, the absorbed nicotine was rapidly and extensively metabolized into cotinine. However, nicotine was found to be predominant in cortex and bladder, where nicotinic acetylcholine receptors were expressed for neuronal control of voiding function. Repeated administration of nicotine led to a ca. 3-fold higher accumulation of nicotine than that of cotinine in rat urine. The results of the pharmacokinetic study using the UPLC/ESI-MS method further support the possible involvement of nicotine in increased risk of urinary dysfunction in smokers.

High dose cotinine may induce neural tube defects in a chick embryo model.

AIM: Nicotine is a well-known agent among 4000 chemicals in cigarettes. About 70 to 80% of nicotine is converted to cotinine, a major metabolite. The aim of the present study is to investigate the effect of cotinine on neural tube development in a chick embryo model. MATERIAL AND METHODS: Sixty fertile, specific pathogen free eggs were divided into 6 groups for this study. In the first group, a fixed cotinine concentration for each egg was calculated just to simulate the concentration of a smoker's blood level. A second experimental group was designed at a higher cotinine concentration. Embryos that succeeded to reach Hamburger-Hamilton stage 12 from each group were then embedded into paraffin for permanent sections. These two groups were compared with eggs subjected to vehicle (standard alcohol and ten times more alcohol concentration) and control groups (saline and sham groups). RESULTS: Embryos of the cotinine (regular dose), vehicle and control groups were normal, but embryos subjected to higher cotinine concentrations were malformed at the cranial part of the thoracic neural tube. CONCLUSION: Association of cotinine with neural tube defects was demonstrated in the present study. Cigarette smoking may induce hazardous effects on neural tube development.

The effect of cotinine on telomerase activity in human vascular smooth muscle cells.

AIM: Cotinine, the main stable metabolite of nicotine, has been shown to have a biological half-life approximately 10 times longer than nicotine. It has also been demonstrated to have a powerful effect on vascular smooth muscle cell (VSMC) proliferation. Telomerase activation is known to play an important role in cell viability and proliferation. The purpose of our experiment was to evaluate the effect of cotinine on proliferative potential of vascular smooth muscle cells via its effects on telomerase activity. METHODS: Primary cultures of human VSMC obtained from greater saphenous veins were used in this experiment from 3(rd) to 5(th) passage. Cotinine was added in doses equivalent to plasma levels of cotinine in an active smoker by dissolving, 0.0, 2.88x10(-6), 5.76x10(-6), and 1.44x10(-5) mol/L of cotinine in the media. The number of viable cells was assessed by trypan blue exclusion. The Telomeric Repeat Amplification Protocol (TRAP) was used to detect telomerase activity. TRAP products were detected by ELISA. RESULTS: The mitogenic effect of cotinine in VSMC was observed at 48 hours after treatment. The viable cell numbers were significantly increased (4.0x10(7)) at lower doses of cotinine exposure as compared to untreated cultures (2.5x10(5)). At the concentration of 1.44x10(-5) mol/L, cotinine was cytotoxic to VSMCs. Telomerase activity was detected in all sets of VSMC cultures treated with cotinine (P<0.01). CONCLUSIONS: Cotinine causes abnormal cell proliferation as demonstrated by increased cell numbers and reactivation of telomerase in a dose dependent manner. This study demonstrated cotinine's stimulatory effect on human SMC proliferation in vitro at low doses while high doses of cotinine had a toxic effect. These data correlate with the results of other studies concerning the mitogenic effect of cotinine and telomerase activation during cellular proliferative response.

Polycyclic aromatic hydrocarbon exposure, urinary mutagenicity, and DNA adducts in rubber manufacturing workers.

OBJECTIVES: Several studies have suggested that genotoxic risks might still be present in the contemporary rubber manufacturing industry. Previously, we observed elevated levels of urinary mutagenicity and bladder DNA adducts in rubber workers. Presently, we investigated whether DNA adducts in peripheral blood mononuclear cells (PBMC) and/or urothelial cells may be caused by polycyclic aromatic hydrocarbons or other genotoxic compounds. METHODS: Spot urine samples from 116 rubber manufacturing workers were collected on Sunday and during the workweek (post-shift) to determine 1-hydroxypyrene and mutagenicity levels. For 52 nonsmokers, urothelial cell DNA adducts and PBMC DNA adducts were measured additionally. RESULTS: Urinary 1-hydroxypyrene levels were significantly higher in workweek samples compared with Sunday (P = 0.0001). This increase was not uniform across tasks and only reached statistical significance for the curing department (+99%; P = 0.003). Weekday urinary mutagenicity was significantly increased for mixing (+56%) and curing (+21%) workers when compared with that for Sunday. Total urothelial cell DNA adducts were related to urinary 1-hydroxypyrene (P = 0.021) and mutagenicity (P = 0.027). No significant relationship was found between the adduct levels in PBMC and urothelial cells or between the former and urinary 1-hydroxypyrene or mutagenicity. CONCLUSIONS: Workers in the compounding, mixing, and curing departments were at highest genotoxic risk among rubber manufacturing workers. Increased levels of urinary 1-hydroxypyrene, mutagenicity, and urothelial cell DNA adducts were found in these workers. Urothelial cell and PBMC DNA adducts were not related, hinting possibly to the presence of specific bladder carcinogens in the rubber manufacturing industry.

Cell proliferation kinetics and genotoxicity in lymphocytes of smokers living in Mexico City.

Genotoxicity caused by tobacco smoke was assessed in peripheral blood lymphocytes of smokers living in Mexico City by determining sister chromatid exchange (SCE), cell proliferation kinetics (CPK), replication index (RI) and mitotic index (MI). Nicotine levels, and its major metabolite cotinine, were also estimated in urine samples using gas-chromatography-mass spectrometry to quantify smoking intensity. The outcome of the analysis and the comparison of the 77-smoker group with a non-smoking control group showed that moderate and heavy smokers exhibited significant differences (P < 0.001 and P < 0.05, respectively) in CPK, with an underlying delay in the cellular cycle; similarly, RI was significantly different in these groups (P < 0.001 and P < 0.0001, respectively). There were significant correlations (P < 0.05) between age and number of years the subject had been smoking, as well as between RI and nicotine and cotinine levels and between CPK (M1, M2 and M3) and nicotine and cotinine levels. Smokers were classified for the analysis according to the nicotine levels (it is in relation to number of cigarettes smoked per day) found in urine (ng/mL) as: light (10-250), moderate (251-850) and heavy (851-4110). Significant differences in CPK were found (P < 0.05) between moderate and heavy smokers and non-smokers. Significant differences in RI were found between moderate (P < 0.001) and heavy smokers (P < 0.0001) and non-smokers, but not for the light smoking group. MI was determined in 57 of the smokers, whereas SCE frequency was only recorded in 34 smokers. Both parameters yielded no significant differences, nor correlations with any of the assessed variables. In conclusion, cytokinetic and cytostatic effects were mainly detected in heavy and moderate smokers. Cell cycle delay and RI decrease were found in all ;healthy' smokers. The nicotine and cotinine exposure (causing oxidative damage to DNA) may have implications in the decrease in cell replication due to direct damage to DNA and/or a decrease in the DNA repair mechanisms. Alternatively, nicotine and cotinine may possibly induce apoptosis.

[Determination of nicotine and cotinine in human biological materials and their significance in toxicological studies]. / Problematyka oznaczania nikotyny i kotyniny w ludzkim materiale biologicznym w aspekcie badan toksykologicznych.

The aim of this study was the preparation of reliable procedure of the determination of nicotine and cotinine both in classic (serum, urine) and alternative biological materials (hair, saliva) and evaluation of their significance for clinical and forensic toxicology. Biological material samples (blood, urine, saliva) were taken from patients after Percutaneous Trans-luminal Coronary Angioplasty (PTCA). The determination of cotinine and nicotine concentration in the biological material should be optimized depending on the aim of analysis. Liquid-liquid extraction procedure and high performance liquid chromatography HPLC/UV-DAD are reliable, specific and relatively cheap. Serum and saliva are valuable biological materials which allow to determine temporary nicotine and cotinine content on the similar level of concentrations. In the near future it will be able to replace blood with saliva sample because of an easy and non-invasive way of sampling. Evaluation of cotinine concentration in urine allows to distinguish the passive from the active tobacco smokers. Hair analysis allows to control a nicotine abstinence as well as a long-term evaluation of the history of smoking. However usage of hair is limited because of difficulty with sampling. Interpretation of results in analysis of alternative materials (hair, saliva) pose a problem because of lack of sampling standardization and lack of standardization of final analysis method.